This invention generally relates to illumination of moving objects or particles for purposes of analysis and detection, and more specifically, to an apparatus and method for increasing the amount of incident light upon these objects to increase scattered, fluorescent, and other signals from moving objects, such as cells, and for detecting the presence and composition of Fluorescence In-Situ Hybridization (FISH) probes within cells.
There are a number of biological and medical applications that are currently impractical due to limitations in cell and particle analysis technology. Examples of such biological applications include battlefield monitoring of known airborne toxins, as well as the monitoring of cultured cells to detect the presence of both known and unknown toxins. Medical applications include non-invasive prenatal genetic testing and routine cancer screening via the detection and analysis of rare cells (i.e., cells with low rates of occurrence) in peripheral blood. All of these applications require an analysis system with the following principal characteristics:
1. the ability to carry out high-speed measurements;
2. the ability to process very large samples;
3. high spectral resolution and bandwidth;
4. good spatial resolution;
5. high sensitivity; and
6. low measurement variation.
In prenatal testing, the target cells are fetal cells that cross the placental barrier into the mother""s blood stream. In cancer screening, the target cells are sloughed into the blood stream from nascent cancerous tumors. In either case, the target cells may be present in the blood at concentrations of one to five target cells per billion blood cells. This concentration yields only 20 to 100 cells in a typical 20 ml blood sample. In these applications, as well as others, it is imperative that the signal derived in response to the cells be as strong as possible to provide distinct features with which to discriminate the target cells from other artifacts in the sample.
It would be desirable to increase the amount of light incident upon objects in a sample compared to prior art systems, thereby increasing the signal-to-noise ratio (SNR) of a processing system, improving measurement consistency, and thus, increasing the discrimination abilities of the system. A spectral imaging cell analysis system is described in a pending commonly assigned U.S. Pat. No. 6,249,341 and entitled, xe2x80x9cImaging And Analyzing Parameters Of Small Moving Objects Such As Cells,xe2x80x9d the drawings and disclosure of which are hereby specifically incorporated herein by reference. This previously filed application describes one approach that is applicable to imaging. It would also be desirable to obtain many of the benefits disclosed in the above-referenced copending application in non-imaging flow cytometers that employ photomultiplier tube (PMT) detectors and any other system that relies on the illumination of objects within a cavity. Depending upon the configuration, substantial benefits should be obtained by increasing the amount of light incident upon an object by as much as a factor of ten or more. Such an increase in the amount of light would enable the use of low power continuous wave (CW) and pulsed lasers in applications that would otherwise require the use of more expensive high power lasers. However, if high power lasers are used for a light source, a processing system should yield higher measurement consistency, higher system throughput, greater illumination uniformity, and other benefits than has been possible with prior systems.
It is a goal in the design of fluorescence instruments to achieve photon-limited performance. When photon-limited performance is achieved, noise sources in the instrument are reduced to insignificance relative to the inherent statistical variation of photon arrivals at the detector. A good example of photon-limited design is found in non-imaging flow cytometers. The PMT detectors employed in these instruments can amplify individual photons thousands of times with very fast rise times.
Non-imaging cytometers take advantage of the PMT""s characteristics to achieve photon-limited performance by making the illuminated area as small as possible. Decreasing the laser spot size reduces the amount of time required for an object to traverse a field of view (FOV) of the detectors. The reduced measurement time, in turn, reduces the integrated system noise, but does not reduce the signal strength of the object. The signal strength remains constant because the reduced signal integration time is balanced by the increased laser intensity in the smaller spot. For example, if the FOV in the axis parallel to flow is decreased by a factor of two, an object""s exposure time will decrease by a factor of two, but the intensity at any point in that FOV will double, so the integrated photon exposure will remain constant.
The reduced noise and constant signal strength associated with a reduced FOV increases the SNR of the non-imaging cytometer up to a point. Beyond that point, further reductions in the FOV will fail to improve the SNR because the dominant source of variation in the signal becomes the inherently stochastic nature of the signal. Photonic signals behave according to Poisson statistics, implying that the variance of the signal is equal to the mean number of photons. Once photon-limited performance is achieved in an instrument, the only way to significantly improve performance is to increase the number of photons that reach the detector.
A common figure of merit used in flow cytometry is the coefficient of variation (CV), which equals the standard deviation of the signal over many measurements divided by the mean of the signal. Photon noise, as measured by the CV, increases as the mean number of photons decreases. For example, if the mean number of photons in a measurement period is four, the standard deviation will be two photons and the CV will be 50%. If the mean number of photons drops to one, the standard deviation will be one and the CV will be 100%. Therefore, to improve (i.e., decrease) the CV, the mean number of photons detected during the measurement interval must be increased. One way to increase the number of photons striking the detector is to increase photon collection efficiency. If an increase in photon collection efficiency is not possible, an alternative is to increase the number of photons emitted from the object during the measurement interval. Accordingly, it would be beneficial to provide a system in which illumination light incident on an object but not absorbed or scattered is recycled and redirected to strike the object multiple times, thereby increasing photon emission from the object.
In the case of a conventional imaging flow cytometer, such as that disclosed in U.S. Pat. No. 5,644,388, a frame-based charge-coupled device (CCD) detector is used for signal detection as opposed to a PMT. In this system, the field of view along the axis of flow is approximately ten times greater than that in PMT-based flow cytometers. In order to illuminate the larger field of view, the patent discloses a commonly used method of illumination in flow cytometry, in which the incident light is directed at the stream of particles in a direction orthogonal to the optic axis of the light collection system. The method disclosed in the patent differs slightly from conventional illumination in that a highly elliptical laser spot is used, with the longer axis of the ellipse oriented along the axis of flow. As a result of this configuration, the entire FOV can be illuminated with laser light. Given that a laser is used, the intensity profile across the illuminated region has a Gaussian profile along the axis of flow. Therefore, objects at either end of the field of view will have a lower intensity of illumination light. Unlike a non-imaging flow cytometer, the light collection process disclosed in this patent does not continue for the duration of the full traversal of the FOV. Instead, light is collected from objects at specific regions within the FOV. Object movement during the light collection process is limited to less than one pixel by use of a shutter or pulsed illumination source. As a result, the amount of light collected from an object varies as a function of its position in the field of view, thereby increasing measurement variability. In order to partially mitigate this variation, the illumination spot may be sized so that it substantially overfills the FOV to use an area of the Gaussian distribution near the peak where the intensity variation is minimized. However, this approach has the undesired effect of reducing the overall intensity of illumination, or photon flux, by spreading the same amount of laser energy over a significantly larger area. The end result of reducing photon flux is a reduction in the SNR.
Accordingly, it will be apparent that an improved technique is desired to improve the SNR and measurement consistency of an instrument by increasing photon emission from the object and improving the uniformity of illumination. It is expected that such a technique will also have applications outside of cell analysis systems and can be implemented in different configurations to meet the specific requirements of disparate applications of the technology.
The present invention is directed to correcting beam misalignments in a multipass cavity illumination system that is adapted to increase the amount of signal emitted from an object to increase the SNR and to improve the measurement consistency of devices in which the present invention is applied.
In general, a multipass cavity is formed by the placement of two mirrors on either side of a moving stream of objects. A light collection system is disposed substantially orthogonal to a plane extending through the mirrors and the stream. The light collection system is configured to collect light over a predefined angle and within a predefined region or field of view between the mirrors. Accordingly, the light collection system collects light that is scattered or emitted from objects as they traverse the space between the mirrors. The scattered or emitted light that is collected is directed onto a detector.
A light from a light source is directed through the stream of moving objects in a direction nearly orthogonal to the stream of objects but slightly inclined in the plane that extends through the mirrors and the stream. With cells and most other objects, only a small fraction of the incident light interacts with the objects via absorbance or scatter. The rest of the light passes through the stream, and is then redirected by reflection from a surface back into the stream of moving objects. The light leaves the reflecting surface at a reflected angle that is equal to an incident angle of the light. Due to the reflection angle and the distance between the stream and the first surface, the light intersects the stream on the second pass at a position that is displaced from that at which the light passed though the stream on its initial pass. The light continues through the stream and is redirected by a second surface on the other side of the stream, which is substantially parallel to the first surface, back towards the stream. Again, as a result of the reflection angle and the distance between the second surface and the stream, the light passes through the stream on the third pass at a position that is displaced from that of the second pass. The reflection of the light through the stream continues a plurality of times until the light has traversed a distance along the direction in which the stream is flowing that is substantially equal to the collected field of view of the light collection system. At this point, the light is no longer reflected back through the stream, but is preferably caused to exit the illumination system.
It should be understood that most of the light that passes through the stream is largely unimpeded by the stream or objects in the stream. Therefore, upon subsequent passes, substantial light remains to intercept the object or objects in the stream. By xe2x80x9crecyclingxe2x80x9d light in this manner, the light that would normally be wasted is employed to illuminate the object each time the object passes through the light. Consequently, the SNR of the instrument is substantially improved by increasing the amount of scattered and/or emitted light that is incident on the detector.
Beam misalignment in a multipass cavity has four degrees of freedom, position in the horizontal (X) and vertical (Y) directions and angle in the vertical and horizontal axes, termed tip and tilt respectively. Misalignments in directions along the beam axis or around the beam axis have little affect on the performance of the cavity. However, angular or positional errors lateral to the propagation direction of the beam can dramatically affect performance. The present invention provides apparatus and methods to easily measure and adjust these errors in an independent fashion for each degree of freedom, in order to make beam steering corrections and maintain optical alignment. The present invention of an active cavity beam detection and alignment system allows this to be accomplished in an automated, closed-loop feedback control system.